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Abstract:

The present invention relates to a plant, which is resistant to a
pathogen of viral, bacterial, fungal or oomycete origin, wherein the
plant has an increased homoserine level as compared to a plant that is
not resistant to the said pathogen, in particular organisms of the phylum
Oomycota. The invention further relates to a method for obtaining a
plant, which is resistant to a pathogen of viral, bacterial, fungal or
oomycete origin, comprising increasing the endogenous homoserine level in
the plant.

Claims:

1. An isolated plant which is resistant to a pathogen, wherein the plant
has an increased endogenous L-homoserine level as compared to a plant
that is not resistant to said pathogen, wherein said plant is selected
from the group consisting of cucumber, grape, and tomato, and wherein
when said plant is cucumber, said pathogen is Pseudoperonospora cubensis
and said cucumber plant has a mutation in the homoserine kinase gene of
SEQ ID NO: 105 lowering the homoserine kinase activity of SEQ ID NO: 106;
wherein when said plant is grape, said pathogen is Plasmopara viticola
and said grape plant has a mutation in the homoserine kinase gene of SEQ
ID NO: 103 lowering the homoserine kinase activity of SEQ ID NO: 104; and
wherein when said plant is tomato, said pathogen is Phytophthora
infestans and said tomato plant has a mutation in the homoserine kinase
gene of SEQ ID NO: 109 lowering the homoserine kinase activity of SEQ ID
NO: 110.

2. The plant of claim 2, wherein the mutation in the homoserine kinase
gene leads to an ammo acid substitution in the encoded protein.

3. A method for obtaining a plant which is resistant to a pathogen,
wherein the plant has an increased endogenous L-homoserine level as
compared to a plant that is not resistant to said pathogen, wherein said
plant is selected from the group consisting of cucumber, grape, and
tomato, the method comprising: increasing the endogenous L-homoserine
level in a cucumber plant by a mutation in the homoserine kinase gene of
SEQ ID NO: 105 lowering the homoserine kinase activity of SEQ ID NO: 106
or reducing the expression of SEQ ID NO: 105 to produce a cucumber plant
which is resistant to Pseudoperonospora cubensis; or increasing the
endogenous L-homoserine level in a grape plant by a mutation in the
homoserine kinase gene of SEQ ID NO: 103 lowering the homoserine kinase
activity of SEQ ID NO: 104 or reducing the expression of SEQ ID NO: 103
to produce a grape plant which is resistant to Plasmopara viticola; or
increasing the endogenous L-homoserine level in a tomato plant by a
mutation in the homoserine kinase gene of SEQ ID NO: 109 lowering the
homoserine kinase activity of SEQ ID NO: 110 or reducing the expression
of SEQ ID NO: 109 to produce a tomato plant which is resistant to
Phytophthora infestans.

4. The method of claim 3, wherein the mutation results in one or more
amino acid changes that lead to a lower homoserine kinase activity.

5. The method of claim 3, wherein the mutation is effected by mutagenic
treatment of the cucumber plant, grape plant, or tomato plant.

6. The method of claim 5, wherein the mutagenic treatment is effected
with a mutagen or radiation.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of copending U.S.
patent application Ser. No. 13/545,853, filed Jul. 10, 2012, which is a
divisional application of U.S. patent application Ser. No. 12/092,253,
filed Dec. 19, 2008, and issued as U.S. Pat. No. 8,237,019, which is a
U.S. National Phase application filed under 35 U.S.C. §371 claiming
priority to PCT Application No. PCT/EP2006/010535, filed Nov. 1, 2006 and
which claims priority to PCT Application No. PCT/EP2005/011718, filed
Nov. 1, 2005, each of which is incorporated herein in reference in their
entirety.

[0002] The Sequence Listing associated with this application is filed in
electronic format via EFS-Web and is hereby incorporated by reference
into the specification in its entirety. The name of the text file
containing the Sequence Listing is 123498_ST25.txt. The size of the text
file is 90,740 bytes, and the text file was created on Dec. 5, 2012.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to disease resistant plants, in
particular plants resistant to organisms of the phylum Oomycota, the
oomycetes. The invention further relates to plant genes conferring
disease resistance and methods of obtaining such disease resistant plants
for providing protection to Oomycota pathogens.

[0004] Resistance of plants to pathogens has been extensively studied, for
both pathogen specific and broad resistance. In many cases resistance is
specified by dominant genes for resistance. Many of these race-specific
or gene-for-gene resistance genes have been identified that mediate
pathogen recognition by directly or indirectly interacting with
avirulence gene products or other molecules from the pathogen. This
recognition leads to the activation of a wide range of plant defense
responses that arrest pathogen growth.

[0005] In plant breeding there is a constant struggle to identify new
sources of mostly monogenic dominant resistance genes. In cultivars with
newly introduced single resistance genes, protection from disease is
often rapidly broken, because pathogens evolve and adapt at a high
frequency and regain the ability to successfully infect the host plant.
Therefore, the availability of new sources of disease resistance is
highly needed.

[0006] Alternative resistance mechanisms act for example through the
modulation of the defense response in plants, such as the resistance
mediated by the recessive mlo gene in barley to the powdery mildew
pathogen Blumeria graminis f. sp. hordei. Plants carrying mutated alleles
of the wildtype MLO gene exhibit almost complete resistance coinciding
with the abortion of attempted fungal penetration of the cell wall of
single attacked epidermal cells. The wild type MLO gene thus acts as a
negative regulator of the pathogen response. This is described in
WO9804586.

[0007] Other examples are the recessive powdery mildew resistance genes,
found in a screen for loss of susceptibility to Erysiphe cichoracearum.
Three genes have been cloned so far, named PMR6, which encodes a pectate
lyase-like protein, PMR4 which encodes a callose synthase, and PMR5 which
encodes a protein of unknown function. Both mlo and pmr genes appear to
specifically confer resistance to powdery mildew and not to oomycetes
such as downy mildews.

[0008] Broad pathogen resistance, or systemic forms of resistance such as
SAR, has been obtained by two main ways. The first is by mutation of
negative regulators of plant defense and cell death, such as in the cpr,
lsd and acd mutants of Arabidopsis. The second is by transgenic
overexpression of inducers or regulators of plant defense, such as in
NPR1 overexpressing plants.

[0009] The disadvantage of these known resistance mechanisms is that,
besides pathogen resistance, these plants often show detectable
additional and undesirable phenotypes, such as stunted growth or the
spontaneous formation of cell death.

[0010] It is an object of the present invention to provide a form of
resistance that is broad, durable and not associated with undesirable
phenotypes.

[0011] In the research that led to the present invention, an Arabidopsis
thaliana mutant screen was performed for reduced susceptibility to the
downy mildew pathogen Hyaloperonospora parasitica. EMS-mutants were
generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight
downy mildew resistant (dmr) mutants were analysed in detail,
corresponding to 6 different loci. Microscopic analysis showed that in
all mutants H. parasitica growth was severely reduced. Resistance of
dmr3, dmr4 and dmr5 was associated with constitutive activation of plant
defence. Furthermore, dmr3 and dmr4, but not dmr5, were also resistant to
Pseudomonas syringae and Golovinomyces orontii.

[0012] In contrast, enhanced activation of plant defense was not observed
in the dmr1, dmr2, and dmr6 mutants. The results of this research have
been described in Van Damme et al. (2005) Molecular Plant-Microbe
Interactions 18(6) 583-592. This article does however not disclose the
identification and characterization of the DMR genes.

BRIEF SUMMARY OF THE INVENTION

[0013] According to the present invention it was now found that DMR1 is
the gene encoding homoserine kinase (HSK). For Arabidopsis five different
mutant dmr1 alleles have been sequenced each leading to a different amino
acid change in the HSK protein. HSK is a key enzyme in the biosynthesis
of the amino acids methionine, threonine and isoleucine and is therefore
believed to be essential. The various dmr1 mutants show defects in HSK
causing the plants to accumulate homoserine The five different alleles
show different levels of resistance that correlate to different levels of
homoserine accumulation in the mutants.

[0014] The present invention thus provides a plant, which is resistant to
a pathogen of viral, bacterial, fungal or oomycete origin, characterized
in that the plant has an altered homoserine level as compared to a plant
that is not resistant to the said pathogen.

[0016] The resistance is based on an altered level of homoserine in
planta. More in particular, the resistance is based on an increased level
of homoserine in planta. Such increased levels can be achieved in various
ways.

[0017] First, homoserine can be provided by an external source. Second,
the endogenous homoserine level can be increased. This can be achieved by
lowering the enzymatic activity of the homoserine kinase gene which leads
to a lower conversion of homoserine and thus an accumulation thereof.
Alternatively, the expression of the homoserine kinase enzyme can be
reduced. This also leads to a lower conversion of homoserine and thus an
accumulation thereof. Another way to increase the endogenous homoserine
level is by increasing its biosynthesis via the aspartate pathway.
Reducing the expression of the homoserine kinase gene can in itself be
achieved in various ways, either directly, such as by gene silencing, or
indirectly by modifying the regulatory sequences thereof or by
stimulating repression of the gene.

[0018] Modulating the HSK gene to lower its activity or expression can be
achieved at various levels. First, the endogenous gene can be directly
mutated. This can be achieved by means of a mutagenic treatment.
Alternatively, a modified HSK gene can be brought into the plant by means
of transgenic techniques or by introgression, or the expression of HSK
can be reduced at the regulatory level, for example by modifying the
regulatory sequences or by gene silencing.

[0019] In one embodiment of the invention, an increase (accumulation) in
homoserine level in the plant is achieved by administration of homoserine
to the plant. This is suitably done by treating plants with L-homoserine,
e.g. by spraying or infiltrating with a homoserine solution.

[0020] Treatment of a plant with exogenous homoserine is known from
WO00/70016. This publication discloses how homoserine is applied to a
plant resulting in an increase in the phenol concentration in the plant.
The publication does not show that plants thus treated are resistant to
pathogens. In fact, WO00/70016 does not disclose nor suggest that an
increase in endogenous homoserine would lead to pathogen resistance.

[0022] In one embodiment, the increased endogenous production is the
result of a reduced endogenous HSK gene expression thus leading to a less
efficient conversion of homoserine into phospho-homoserine and the
subsequent biosynthesis of methionine and threonine. This reduced
expression of HSK is for example the result of a mutation in the HSK gene
leading to reduced mRNA or protein stability.

[0023] In another embodiment reduced expression can be achieved by
downregulation of the HSK gene expression either at the transcriptional
or the translational level, e.g. by gene silencing or by mutations in the
regulatory sequences that affect the expression of the HSK gene. An
example of a method of achieving gene silencing is by means of RNAi.

[0024] In a further embodiment the increase in endogenous homoserine level
can be obtained by inducing changes in the biosynthesis or metabolism of
homoserine. In a particular embodiment this is achieved by mutations in
the HSK coding sequence that result in a HSK protein with a reduced
enzymatic activity thus leading to a lower conversion of homoserine into
phospho-homoserine. Another embodiment is the upregulation of genes in
the aspartate pathway causing a higher production and thus accumulation
of L-homoserine in planta.

[0026] Table 2 shows the Genbank accession numbers and GenInfo identifiers
of the Arabidopsis HSK mRNA and orthologous sequences from other plant
species.

[0027] FIG. 2 shows the percentage of conidiophore formation by two
Hyaloperonospora parasitica isolates, Cala2 and Waco9, on the mutants
dmr1-1, dmr1-2, dmr1-3 and dmr1-4 and the parental line, Ler eds1-2, 7
days post inoculation. The conidiophores formed on the parental line were
set to 100%.

[0028] FIG. 3 is a graphic overview of the three major steps in the
cloning of DMR1. a) Initial mapping of dmr1 resulted in positioning of
the locus on the lower arm of chromosome 2 between positions 7.42 and
7.56 Mb. Three insert/deletion (INDEL) markers were designed (position of
the markers F6P23, T23A1 and F5J6 is indicated by the black lines). These
markers were used to identify recombinants from several 100 segregating
F2 and F3 plants. Primer sequences of these INDEL markers and additional
markers to identify the breakpoints in the collected recombinants is
presented in table 3. b) One marker, At2g17270 (indicated by the grey
line), showed the strongest linkage with resistance. The dmr1 locus could
be further delimited to a region containing 8 genes, at2g17250-at2g17290.
The eight genes were amplified and sequenced to look for mutations in the
coding sequences using the primers described in table 4. DNA sequence
analysis of all 8 candidate genes led to the discovery of point mutations
in the At2g17265 gene in all 5 dmr1 mutants. c) Each dmr1 mutant has a
point mutation at a different location in the At2g17265 gene, which
encodes homoserine kinase.

[0029] FIG. 4 shows a schematic drawing of the HSK coding sequence and the
positions and nucleotide substitutions of the 5 different dmr1 mutations
in the HSK coding sequence (the nucleotide positions, indicated by the
black triangles, are relative to the ATG start codon which start on
position i). The 5'UTR and 3'UTR are shown by light grey boxes. Below the
nucleotide sequence the protein sequence is shown. The HSK protein
contains a putative transit sequence for chloroplast targeting (dark grey
part). The amino acid changes resulting from the 5 dmr1 mutations are
indicated at their amino acid (aa) position number (black triangles) in
the HSK protein.

[0030] FIG. 5 shows the position of the homoserine kinase enzyme in the
aspartate pathway for the biosynthesis of the amino acids threonine,
methionine and isoleucine.

[0031] FIG. 6 shows the number of conidiophores per Ler eds 1-2 seedlings
5 days post inoculation with two different isolates of H. parasitica,
Waco9 and Cala2. The inoculated seedlings were infiltrated with dH2O,
D-homoserine (5 mM) or L-homoserine (5 mM) at 3 days post inoculation
with the pathogen. Seedlings treated with L-homoserine show a complete
absence of conidiophore formation and are thus resistant.

[0032] FIG. 7 shows the growth and development of H. parasitica in
seedlings treated with water, D-homoserine (5 mM), or L-homoserine (5 mM)
as analysed by microscopy of trypan blue stained seedlings.

[0041] This invention is based on research performed on resistance to
Hyaloperonospora parasitica in Arabidopsis but is a general concept that
can be more generally applied in plants, in particular in crop plants
that are susceptible to infections with pathogens, such as Oomycota.

[0042] The invention is suitable for a large number of plant diseases
caused by oomycetes such as, but not limited to, Bremia lactucae on
lettuce, Peronospora farinosa on spinach, Pseudoperonospora cubensis on
members of the Cucurbitaceae family, e.g. cucumber, Peronospora
destructor on onion, Hyaloperonospora parasitica on members of the
Brasicaceae family, e.g. cabbage, Plasmopara viticola on grape,
Phytophthora infestans on tomato and potato, and Phytophthora sojae on
soybean.

[0043] The homoserine level in these other plants can be increased with
all techniques described above. However, when the modification of the HSK
gene expression in a plant is to be achieved via genetic modification of
the HSK gene or via the identification of mutations in the HSK gene, and
the gene is not yet known it must first be identified. To generate
pathogen-resistant plants, in particular crop plants, via genetic
modification of the HSK gene or via the identification of mutations in
the HSK gene, the orthologous HSK genes must be isolated from these plant
species. Orthologs are defined as the genes or proteins from other
organisms that have the same function.

[0044] Various methods are available for the identification of orthologous
sequences in other plants.

[0045] A method for the identification of HSK orthologous sequences in a
plant species, may for example comprise identification of homoserine
kinase ESTs of the plant species in a database; designing primers for
amplification of the complete homoserine kinase transcript or cDNA;
performing amplification experiments with the primers to obtain the
corresponding complete transcript or cDNA; and determining the nucleotide
sequence of the transcript or cDNA.

[0046] Suitable methods for amplifying the complete transcript or cDNA in
situations where only part of the coding sequence is known are the
advanced PCR techniques 5'RACE, 3'RACE, TAIL-PCR, RLM-RACE and vectorette
PCR.

[0047] Alternatively, if no nucleotide sequences are available for the
plant species of interest, primers are designed on the HSK gene of a
plant species closely related to the plant of interest, based on
conserved domains as determined by multiple nucleotide sequence
alignment, and used to PCR amplify the orthologous sequence. Such primers
are suitably degenerate primers.

[0048] Another reliable method to assess a given sequence as being a HSK
ortholog is by identification of the reciprocal best hit. A candidate
orthologous HSK sequence of a given plant species is identified as the
best hit from DNA databases when searching with the Arabidopsis HSK
protein or DNA sequence, or that of another plant species, using a Blast
programme. The obtained candidate orthologous nucleotide sequence of the
given plant species is used to search for homology to all Arabidopsis
proteins present in the DNA databases (e.g. at NCBI or TAIR) using the
BlastX search method. If the best hit and score is to the Arabidopsis HSK
protein, the given DNA sequence can be described as being an ortholog, or
orthologous sequence.

[0049] HSK is encoded by a single gene in Arabidopsis and rice as deduced
from the complete genome sequences that are publicly available for these
plant species. In most other plant species tested so far, HSK appears to
be encoded by a single gene, as determined by the analysis of mRNA
sequences and EST data from public DNA databases, except for potato,
tobacco and poplar for which two HSK homologs have been identified. The
orthologous genes and proteins are identified in these plants by
nucleotide and amino acid comparisons with the information that is
present in public databases.

[0050] Alternatively, if no DNA sequences are available for the desired
plant species, orthologous sequences are isolated by heterologous
hybridization using DNA probes of the HSK gene of Arabidopsis or another
plant or by PCR methods, making use of conserved domains in the HSK
coding sequence to define the primers. For many crop species, partial HSK
mRNA sequences are available that can be used to design primers to
subsequently PCR amplify the complete mRNA or genomic sequences for DNA
sequence analysis.

[0051] In a specific embodiment the ortholog is a gene of which the
encoded protein shows at least 50% identity with the Arabidopsis HSK
protein or that of other plant HSK proteins. In a more specific
embodiment the homology is at least 55%, more specifically at least 60%,
even more specifically at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95% or at least 99%.

[0052] After orthologous HSK sequences are identified, the complete
nucleotide sequence of the regulatory and coding sequence of the gene is
identified by standard molecular biological techniques. For this, genomic
libraries of the plant species are screened by DNA hybridization or PCR
with probes or primers derived from a known homoserine kinase gene, such
as the above described probes and primers, to identify the genomic clones
containing the HSK gene. Alternatively, advanced PCR methods, such as RNA
Ligase Mediated RACE (RLM-RACE), can be used to directly amplify gene and
cDNA sequences from genomic DNA or reverse-transcribed mRNA. DNA
sequencing subsequently results in the characterization of the complete
gene or coding sequence.

[0053] Once the DNA sequence of the gene is known this information is used
to prepare the means to modulate the expression of the homoserine kinase
gene in anyone of the ways described above.

[0054] More in particular, to achieve a reduced HSK activity the
expression of the HSK gene can be down-regulated or the enzymatic
activity of the HSK protein can be reduced by amino acid substitutions
resulting from nucleotide changes in the HSK coding sequence.

[0055] In a particular embodiment of the invention, downregulation of HSK
gene expression is achieved by gene-silencing using RNAi. For this,
transgenic plants are generated expressing a HSK anti-sense construct, an
optimized micro-RNA construct, an inverted repeat construct, or a
combined sense-anti-sense construct, so as to generate dsRNA
corresponding to HSK that leads to gene silencing.

[0056] In an alternative embodiment, one or more regulators of the HSK
gene are downregulated (in case of transcriptional activators) by RNAi.

[0057] In another embodiment regulators are upregulated (in case of
repressor proteins) by transgenic overexpression. Overexpression is
achieved in a particular embodiment by expressing repressor proteins of
the HSK gene from a strong promoter, e.g. the 35S promoter that is
commonly used in plant biotechnology.

[0058] The downregulation of the HSK gene can also be achieved by
mutagenesis of the regulatory elements in the promoter, terminator
region, or potential introns. Mutations in the HSK coding sequence in
many cases lead to amino acid substitutions or premature stop codons that
negatively affect the expression or activity of the encoded HSK enzyme.

[0059] These and other mutations that affect expression of HSK are induced
in plants by using mutagenic chemicals such as ethyl methane sulfonate
(EMS), by irradiation of plant material with gamma rays or fast neutrons,
or by other means. The resulting nucleotide changes are random, but in a
large collection of mutagenized plants the mutations in the HSK gene can
be readily identified by using the TILLING (Targeting Induced Local
Lesions IN Genomes) method (McCallum et al. (2000) Targeted screening for
induced mutations. Nat. Biotechnol. 18, 455-457, and Henikoff et al.
(2004) TILLING. Traditional mutagenesis meets functional genomics. Plant
Physiol. 135, 630-636). The principle of this method is based on the PCR
amplification of the gene of interest from genomic DNA of a large
collection of mutagenized plants in the M2 generation. By DNA sequencing
or by looking for point mutations using a single-strand specific
nuclease, such as the CEL-I nuclease (Till et al. (2004) Mismatch
cleavage by single-strand specific nucleases. Nucleic Acids Res. 32,
2632-2641) the individual plants that have a mutation in the gene of
interest are identified.

[0060] By screening many plants, a large collection of mutant alleles is
obtained, each giving a different effect on gene expression or enzyme
activity. The gene expression or enzyme activity can be tested by
analysis of HSK transcript levels (e.g. by RT-PCR), quantification of HSK
protein levels with antibodies or by amino acid analysis, measuring
homoserine accumulation as a result of reduced HSK activity. These
methods are known to the person skilled in the art.

[0061] The skilled person can use the usual pathogen tests to see if the
homoserine accumulation is sufficient to induce pathogen resistance.

[0062] Plants with the desired reduced HSK activity or expression are then
back-crossed or crossed to other breeding lines to transfer only the
desired new allele into the background of the crop wanted.

[0063] The invention further relates to mutated HSK genes encoding HSK
proteins with a reduced enzymatic activity. In a particular embodiment,
the invention relates to the dmr1 alleles dmr1-1, dmr1-2, dmr1-3, dmr1-4
and dmr1-5.

[0065] The present invention demonstrates that plants having an increased
homoserine level show resistance to pathogens, in particular of oomycete
origin. With this knowledge the skilled person can actively modify the
HSK gene by means of mutagenesis or transgenic approaches, but also
identify so far unknown natural variants in a given plant species that
accumulate homoserine or that have variants of the HSK gene that lead to
an increase in homoserine, and to use these natural variants according to
the invention.

[0066] In the present application the terms "homoserine kinase" and "HSK"
are used interchangeably.

[0067] The present invention is illustrated in the following examples that
are not intended to limit the invention in any way. In the examples
reference is made to the following figures.

EXAMPLES

Example 1

Characterization of the Gene Responsible for Pathogen Resistance in dmr
Mutants

[0068] Van Damme et al., 2005, supra disclose four mutants, dmr1-1,
dmr1-2, dmr1-3 and dmr1-4 that are resistant to H. parasitica. The level
of resistance can be examined by counting conidiophores per seedling leaf
seven day post inoculation with the H. parasitica Cala2 isolate
(obtainable from Dr. E. Holub (Warwick HRI, Wellesbourne, UK or Dr. G.
Van den Ackerveken, Department of Biology, University of Utrecht,
Utrecht, NL). For the parental line, Ler eds1-2 (Parker et al., 1996.
Plant Cell 8:2033-2046), which is highly susceptible, the number of
conidiophores is set at 100%. The reduction in conidiophore formation on
the infected dmr1 mutants compared to seedlings of the parental line is
shown in FIG. 2.

[0069] According to the invention, the gene responsible for resistance to
H. parasitaca in the dmr1 mutants of van Damme el al., 2005, supra has
been cloned by a combination of mapping and sequencing of candidate
genes.

[0070] DMR1 was isolated by map-based cloning. The dmr1 mutants were
crossed to the FN2 Col-0 mutant to generate a mapping population. The FN2
mutant is susceptible to the H. parasitica isolate Cala2, due to a fast
neutron mutation in the RPP7A gene (Sinapidou et al., 2004, Plant J.
38:898-909). All 5 dmr1 mutants carry single recessive mutations as the
F1 plants were susceptible, and approximately a quarter of the F2 plants
displayed H. parasitica resistance.

[0071] The DMR1 cloning procedure is illustrated in FIG. 3 and described
in more detail below. The map location of the dmr1 locus was first
determined by genotyping 48 resistant F2 plants to be located on the
lower arm of chromosome 2. From an additional screen for new recombinants
on 650 F2 plants ˜90 F2 recombinant plants between two INDEL
(insertion/deletion) markers on BAC T24112 at 7.2 Mb and BAC F5J6 at 7.56
Mb (according to the TIGR Arabidopsis genome release Version 5.0 of
January 2004) were identified, which allowed to map the gene to a region
containing a contig of 5 BACs.

[0072] The F2 plants were genotyped and the F3 generation was phenotyped
in order to fine map the dmr1 locus. The dmr1 mutation could be mapped to
a ˜130 kb region (encompassing 3 overlapping BAC clones: F6P23,
T23A1, and F5J6) between two INDEL markers located on BAC F6P23, at 7.42
Mb and F5J6 at 7.56 Mb (according to the TIGR Arabidopsis genome release
Version 5.0 of January 2004). This resulted in an area of 30 putative
gene candidates for the dmr1 locus, between the Arabidopsis genes with
the TAIR codes AT2g17060 and AT2g17380. Additionally cleaved amplified
polymorphic sequences (CAPS) markers were designed based on SNPs linked
to genes AT2g17190, AT2g17200, AT2g17270, At2g17300, At2g17310 and
At2g17360 genes.

[0073] Analyses of 5 remaining recombinants in this region with these CAPS
marker data left 8 candidate genes, At2g17230 (NM--127277,
GI:30679913), At2g17240 (NM--127278, GI:30679916), At2g17250
(NM--127279, GI:22325730), At2g17260 (NM--127280, GI:30679922).
At2g17265 (NM--127281, GI:18398362), At2g17270 (NM--127282,
GI:30679927), At2g17280 (NM--127283, GI:42569096), At2g17290
(NM--127284, GI:30679934). Sequencing of all the 8 genes resulted in
the finding of point mutations in the AT2g17265 coding gene in the five
dmr1 alleles: dmr1-1, dmr1-2, dmr1-3, dmr1-4 and dmr1-5, clearly
demonstrating that AT2g17265 is DMR1. FIG. 3 shows a scheme of dnrl with
point mutations of different alleles.

[0075] In Arabidopsis, HSK is encoded by a single gene. At2g17265 (Lee &
Leustek, 1999, Arch. Biochem. Biophys. 372: 135-142). HSK is the fourth
enzyme in the aspartate pathway required for the biosynthesis of the
amino acids methionine, threonine and isoleucine. HSK catalyzes the
phosphorylation of homoserine to homoserine phosphate (FIG. 5).

Example 2

Amino Acid Analysis

[0076] Homoserine phosphate is an intermediate in the production of
methionine, isoleucine and threonine in Arabidopsis. Since homoserine
kinase has a key role in the production of amino acids, free amino acid
levels were determined in the parental line Ler eds1-2 and the four
different dmr1 mutants. For this amino acids from total leaves were
extracted with 80% methanol, followed by a second extraction with 20%
methanol. The combined extracts were dried and dissolved in water. After
addition of the internal standard, S-amino-ethyl-cysteine (SAEC) amino
acids were detected by automated ion-exchange chromatography with post
column ninhydrin derivatization on a JOEL AminoTac JLC-500/V (Tokyo,
Japan).

[0077] Amino acid analysis of four different dmr1 mutants and the parental
line, Ler eds 1-2 showed an accumulation of homoserine in the dmr1
mutants, whereas this intermediate amino acid was not detectable in the
parental line Ler eds1-2. There was no reduction in the level of
methionine, isoleucine and threonine in the dmr1 mutants (Table 1).

Due to the reduced activity of the HSK in the dmr1 mutants, homoserine
accumulates. This effect could be further enhanced by a stronger influx
of aspartate into the pathway leading to an even higher level of
homoserine. The high concentration of the substrate homoserine would
still allow sufficient phosphorylation by the mutated HSK so that the
levels of methionine, isoleucine and threonine are not reduced in the
dmr1 mutants and the parental line, Ler eds1-2 (Table 1).

Example 3

Pathogen Resistance is Achieved by Application of L-Homoserine

[0078] To test if the effect is specific for homoserine the stereo-isomer
D-homoserine was tested. Whole seedlings were infiltrated with water, 5
mM D-homoserine and 5 mM L-homoserine. Only treatment with the natural
amino acid L-homoserine resulted in resistance to H. parasitica.
Seedlings treated with water or D-homoserine did not show a large
reduction in pathogen growth and were susceptible to H. parasitica. The
infiltration was applied to two Arabidopsis accessions, Ler eds1-2 and Ws
eds1-1, susceptible to Cala2 and Waco9, respectively. Conidiophore
formation was determined as an indicator for H. parasitica
susceptibility. Conidiophores were counted 5 days post inoculation with
H. parasitica and 2 days post infiltration with water, D-homoserine or
L-homoserine. (FIG. 6). L-homoserine infiltration clearly results in
reduction of conidiophore formation and H. parasitica resistance. This
was further confirmed by studying pathogen growth in planta by trypan
blue staining of Arabidopsis seedlings. Plants were inoculated with
isolate Cala2. Two days later the plants were treated by infiltration
with water, 5 mM D-homoserine, and 5 mM L-homoserine. Symptoms were
scored at 5 days post inoculation and clearly showed that only the
L-homoserine-infiltrated seedlings showed a strongly reduced pathogen
growth and no conidiophore formation (FIG. 7).

[0079] Microscopic analysis showed that only in L-homoserine treated
leaves the haustoria, feeding structures that are made by H. parasitica
during the infection process, are disturbed. Again it is shown that
increased levels of homoserine in planta lead to pathogen resistance.

Example 4

Identification of HSK Orthologs in Crops

1. Screening of Libraries on the Basis of Sequence Homology

[0080] The nucleotide and amino acid sequences of the homoserine kinase
gene and protein of Arabidopsis thaliana are shown in FIGS. 8 and 9 (SEQ
ID NOs: 99-100).

[0081] Public libraries of nucleotide and amino acid sequences were
compared with the sequences of FIGS. 8 and 9 (SEQ ID NOs: 99-100).

This comparison resulted in identification of the complete HSK coding
sequences and predicted amino acid sequences in Citrus sinensis, Populus
trichocarpa (1), Populus trichocarpa (2), Solanum tuberosum (2), Solanum
tuberosum (1), Nicotiana benthamiana, Ipomnoea nil, Glycine max,
Phaseolus vulgaris, Pinus taeda, Zea mays, and Oryza sativa. The sequence
information of the orthologous proteins thus identified is given in FIG.
1. For many other plant species orthologous DNA fragments could be
identified by BlastX as reciprocal best hits to the Arabidopsis or other
plant HSK protein sequences.

2. Identification of Orthologs by Means of Heterologous Hybridisation

[0082] The HSK DNA sequence of Arabidopsis thaliana as shown in FIG. 8
(SEQ ID NO: 99) is used as a probe to search for homologous sequences by
hybridization to DNA on any plant species using standard molecular
biological methods. Using this method orthologous genes are detected by
southern hybridization on restriction enzyme-digested DNA or by
hybridization to genomic or cDNA libraries. These techniques are well
known to the person skilled in the art. As an alternative probe the HSK
DNA sequence of any other more closely related plant species can be used
as a probe.

3. Identification of Orthologs by Means of PCR

[0083] For many crop species, partial HSK mRNA or gene sequences are
available that are used to design primers to subsequently PCR amplify the
complete cDNA or genomic sequence. When 5' and 3' sequences are available
the missing internal sequence is PCR amplified by a HSK specific 5'
forward primer and 3' reverse primer. In cases where only 5', internal or
3' sequences are available, both forward and reverse primers are
designed. In combination with available plasmid polylinker primers,
inserts are amplified from genomic and cDNA libraries of the plant
species of interest. In a similar way, missing 5' or 3' sequences are
amplified by advanced PCR techniques, 5'RACE, 3'RACE, TAIL-PCR, RLM-RACE
or vectorette PCR.

[0084] As an example the sequencing of the Lactuca sativa (lettuce) HSK
cDNA is provided. From the Genbank EST database at NCBI several Lactuca
HSK ESTs were identified using the tblastn tool starting with the
Arabidopsis HSK amino acid sequence. Clustering and alignment of the ESTs
resulted in a consensus sequence for a 5'HSK fragment and one for a 3'
HSK fragment. To obtain the complete lettuce HSK cDNA the RLM-RACE kit
(Ambion) was used on mRNA from lettuce seedlings. The 5' mRNA sequence
was obtained by using a primer that was designed in the 3'HSK consensus
sequence derived from ESTs (R1S1a: GCCTTCTTCACAGCATCCATTCC--SEQ ID NO: 1)
and the 5'RACE primers from the kit. The 3' cDNA sequence was obtained by
using two primers designed on the 5'RACE fragment (Let3 RACEOut:
CCOTTGCGGTTAATGAGATT--SEQ ID NO: 2, and Let3RACEInn:
TCGTGTTGGTGAATCCTGAA--SEQ ID NO: 3) and the 3'RACE primers from the kit.
Based on the assembled sequence new primers were designed to amplify the
complete HSK coding from cDNA to provide the nucleotide sequence and
derived protein sequence as presented in FIG. 10 (SEQ ID NOs: 101-102). A
similar approach was a used for Solanum lycopersicum (FIG. 14--SEQ ID
NOs: 109-110) and Vitis vinifera (FIG. 11--SEQ ID NOs: 103-104).

[0085] The complete HSK coding sequences from more than 10 different
plants species have been identified from genomic and EST databases. From
the alignment of the DNA sequences, conserved regions in the coding
sequence were selected for the design of degenerate oligonucleotide
primers (for the degenerate nucleotides the abbreviations are according
to the IUB nucleotide symbols that are standard codes used by all
companies synthesizing oligonucleotides, G=Guanine, A=Adenine, T=Thymine,
C=Cytosine, R=A or G, Y=C or T, M=A or C, K=G or T, S=C or G, W=A or T,
B=C or G or T, D=G or A or T, H=A or C or T, V=A or C or G, N=A or C or G
or T).

[0086] The procedure for obtaining internal HSK cDNA sequences of a given
plant species is as follows:

[0087] 1. mRNA is isolated using standard methods,

[0088] 2. cDNA is synthesized using an oligo dT primer and standard
methods,

[0096] Orthologs identified as described in this example can be modified
using well-known techniques to induce mutations that reduce the HSK
expression or activity. Alternatively, the genetic information of the
orthologs can be used to design vehicles for gene silencing. All these
sequences are then used to transform the corresponding crop plants to
obtain plants that are resistant to Oomycota.

Example 5

Reduction of Homoserine Kinase Expression in Arabidopsis by means of RNAi

[0097] The production of HSK silenced lines has been achieved in
Arabidopsis by RNAi. A construct containing two ˜750 bp fragments
of the HSK exon in opposite directions was successfully transformed into
the Arabidopsis Col-0 accession. The transformants were analysed for
resistance to H. parasitica, isolate Waco9. Several transgenic lines were
obtained that confer resistance to H. parasitica. Analysis of HSK
expression and homoserine accumulation confirm that in the transformed
lines the HSK gene is silenced, resulting in resistance to H. parasitica.

Example 6

Mutation of Seeds

[0098] Seeds of the plant species of interest are treated with a mutagen
in order to introduce random point mutations in the genome. Mutated
plants are grown to produce seeds and the next generation is screened for
increased accumulation of homoserine. This is achieved by measuring
levels of the amino acid homoserine, by monitoring the level of HSK gene
expression, or by searching for missense mutations in the HSK gene by the
TILLING method, by DNA sequencing, or by any other method to identify
nucleotide changes.

[0099] The selected plants are homozygous or are made homozygous by
selfing or inter-crossing. The selected homozygous plants with increased
homoserine levels are tested for increased resistance to the pathogen of
interest to confirm the increased disease resistance.

Example 7

Transfer of a Mutated Allele into the Background of a Desired Crop

[0100] Introgression of the desired mutant allele into a crop is achieved
by crossing and genotypic screening of the mutant allele. This is a
standard procedure in current-day marker assistant breeding of crops.